Hydrocoating: a new method for coupling biomolecules to solid phases

Gregorius, Klaus; Mourit­sen, Søren; Elsner, Henrik I.

doi:10.1016/0022-1759(94)00330-y

Cited by 43 publications

(25 citation statements)

References 17 publications

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“…Previously published results [18] and the results presented here document that when peptides are covalently immobilized, recognition of the peptides by antibodies is improved. On the AquaBind microtiter plate, peptides are covalently immobilized in a one step procedure and this makes the AquaBind microtiter plate very useful as a screening tool and in the development of peptide based diagnostics kits.…”

Section: Discussionsupporting

confidence: 67%

“…It was shown previously that the covalent immobilization method is generally superior to the conventional, non-covalent adsorption method with regard to recognition of peptides by antibodies [18]. In this paper, the authors demonstrate that the use of the covalent immobilization method in a peptidebased epitope scan gives a more complete picture of the distribution of linear epitopes in a protein antigen.…”

Section: Discussionmentioning

confidence: 92%

“…AquaBind microtiter plates, containing tresyl-activated dextran molecules produced as previously described [18], were manufactured at M&E Biotech's production facilities. For non-covalent coupling experiments Maxisorp microtiter plates (NUNC, Roskilde, Denmark) were used.…”

Section: Immobilization Of Peptides and Performance Of The Epitope Scanmentioning

confidence: 99%

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A novel microtiter plate based method for identification of B-cell epitopes

Gregorius¹,

Dalum²,

Freisleben³

et al. 1999

J. Peptide Sci.

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A new type of microtiter plate capable of binding biomolecules covalently in a one step procedure was used to map linear B-cell epitopes in two different proteins using a peptide-based solid phase immunoassay. The method was compared with a conventional immobilization method using passive adsorption to microtiter plates. An array of 15-mer peptides, overlapping by five amino acids, representing the entire sequences of ubiquitin and murine tumor necrosis factor-alpha, respectively, was synthesized. The peptides were immobilized covalently using the new, specialized microtiter plates or non-covalently using conventional ELISA microtiter plates of the high binder type. Subsequently, specific antisera to ubiquitin or murine tumor necrosis factor-alpha were added to identify potential linear B-cell epitopes. All peptides, which were recognized on the conventional microtiter plates, were also recognized on the plates with the covalently bound peptides. In addition, the covalent immobilization method revealed epitopes that were not identified using the method for non-covalent binding although the peptides were in fact present on the non-covalent binding surface. The interaction with the hydrophobic surface of the conventional microtiter plate apparently interfered negatively with antibody recognition. The covalently binding microtiter plates described here could be useful for identification of new B-cell epitopes in protein antigens.

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Section: Discussionsupporting

confidence: 67%

Section: Discussionmentioning

confidence: 92%

See 1 more Smart Citation

A novel microtiter plate based method for identification of B-cell epitopes

Gregorius¹,

Dalum²,

Freisleben³

et al. 1999

J. Peptide Sci.

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“…The functional antigen was up to 60%, about 10 times higher than directly coating. Therefore, analytical sensitivity was increased (7)(8)(9). For the anti-PR3 capture ELISA adopted in this study, a plate precoated with a monoclonal antibody was used to capture the antigen.…”

Section: Discussionmentioning

confidence: 99%

Clinical relevance of anti‐PR3 capture ELISA in diagnosing Wegener's granulomatosis

Feng¹,

Liu²,

Li³

et al. 2008

Clinical Laboratory Analysis

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Wegener's granulomatosis (WG) is one type of systemic small vessel vasculitis and antineutrophil cytoplasmic antibodies (ANCA) have become an established diagnostic tool for systemic vasculitis. The sensitivity and specificity of anti-PR3 (proteinase 3) capture enzyme-linked immunosorbent assay (ELI-SA) in diagnosing WG were investigated, as well as the correlation with the indirect immunofluorescence test (IIFT). Sera from 72 patients with WG, 100 disease controls, and 206 healthy blood donors were investigated for anti-PR3 and cytoplasmic ANCA (cANCA) by anti-PR3 classic ELISA, anti-PR3 capture ELISA, and IIFT. The sensitivity of anti-PR3 classic ELISA and capture ELISA in diagnosing WG was 74% and 87.5% separately. The specificity of the two ELISA was identical (100%). For the combination of IIFT with anti-PR3 capture ELISA, the sensitivity for WG patients was up to 91.6%. The sensitivity of anti-PR3 capture ELISA is superior to anti-PR3 classic ELISA, and the correlation between anti-PR3 capture ELISA and IIFT is also more superior. For suspected WG, anti-PR3 capture ELISA and IIFT should be applied in parallel.

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“…Furthermore, the covalent immobilization leaves most of the peptide molecule free from the surface and hence free to interact with, e.g., an antibody (5, 7). The AquaBind microtiter plate has tresyl groups on the surface (10), which react with primary amino and thiol groups, in peptides found in the N-terminus (␣-amine), lysine side chain (-amine), and cysteine side chain, respectively. However, if the lysine and/or cysteine side chains are included in the epitope, the epitope may be chemically modified when binding to the tresylated surface and in the worst case an antibody will not recognize the epitope.…”

mentioning

confidence: 99%